The development of inhibitory antibodies to fVIII (fVIII) is the most significant complication in management of hemophilia A. Moreover, autoantibodies to fVIII in nonhemophiliacs produce the most common autoimmune bleeding disorder involving the coagulation system. In this application, we propose an experimental approach towards developing a comprehensive understanding of the humoral response to fVIII in human and murine hemophilia A. In Aim 1, we will characterize the diversity of anti-human and anti-porcine fVIII B cell epitopes recognized by Hemophilia A mice. We will determine the relative immunodominance of human and porcine fVIII domains using a novel mapping method. This method will be extended to identify all of the non-overlapping epitopes that are targeted by anti-human or anti-porcine fVIII antibodies in the murine Hemophilia A model. In the process, we expect to find important new epitopes and/or common epitopes that are recognized by most or all individuals. In Aim 2, we will characterize the interaction of fVIII with a class of antibodies that bind to both the A1 and A3 domains of human fVIll that we have recently discovered. We hypothesize that these antibodies cross-link fVIII on the B cell surface, which initiates signaling of immunogenic pathways. In Aim 3, we will produce a low immunogenicity fVIII molecule by substituting hypoantigenic porcine sequences identified in the first 2 aims for the corresponding human sequences. Candidate low immunogenicity molecules will be tested in a Hemophilia A mouse model. Our ultimate goal is to identify a candidate low immunogenicity fVIII molecule for comparison to human fVIII in clinical trials in previously untreated patients (PUPs) with hemophilia A and in Hemophilia A gene therapy trials. In Aim 4, we will study the natural history of anti-fVIII antibody development in severe Hemophilia A PUPs using a sensitive anti-fVIII immunoassay and a novel domain specific immunoassay resulting from Aim 1. We anticipate these assays will be useful prognosticators for the development of clinically significant inhibitory antibodies. We intend to test this hypothesis by participating in the Hemophilia Inhibitor PUP Study (HIPS), a prospective natural history cohort study of inhibitor development of PUPs with severe hemophilia A. As an outcome of this project, new therapeutic and diagnostic approaches in the management of Hemophilia A will be produced.